1H, 15N and 13C resonance assignments,secondary structure,and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase

Summary By using fully ¹⁵N- and ¹⁵N/¹³C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific ¹H and ¹⁵N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the ¹³C resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected ¹⁵N and ¹³C experiments. A smaller but significant subset of side-chain ¹H and ¹³C assignments were also determined. In this complex, only one ¹⁵N or ¹³C resonance was detected per ¹⁵N or ¹³C protein nucleus, which indicated a single conformation. Proton-detected ¹³C experiments were also performed with unlabeled DHFR, complexed with ¹³C-7/¹³C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on ¹H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667–9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.     

Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B

Summary A rapid and sensitive 2D approach is presented for measuring amide proton exchange rates and the NOE interaction between amide protons and water. The approach is applicable to uniformly ¹³C/¹⁵N-enriched proteins and can measure magnetization exchange rates in the 0.02 to >20s^–1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying H resonances, followed by NOESY-or ROESY-type transfer to amide protons, which are dispersed by the amide ¹⁵N frequencies in an HSQC-type experiment. Two separate but interleaved experiments, with and without selective inversion of the H₂O resonance, yield quantitative results. The method is demonstrated for a sample of the calcium-binding protein calcineurin B. Results indicate rapid amide exchange for the five calcineurin B residues that are analogous to the five rapidly exchanging residues in the central helix of the homologous protein calmodulin.     

Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

Methods are described to correlate aromatic ¹H ²/¹³C ² or ¹H ¹/¹⁵N ¹ with aliphatic ¹³C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [¹⁵N, ¹H]- and/or [¹³C, ¹H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [¹⁵N, ¹H]-TROSY-H(N)C^ar experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups.     

(H)N(COCA)NH and HN(COCA)NH experiments for 1H-15N backbone assignments in 13C/15N-labeled proteins

Triple resonance HN(COCA)NH pulse sequences for correlating 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins that utilize overlapping coherence transfer periods provide increased sensitivityrelative to pulse sequences that utilize sequential coherence transfer periods. Although theoverlapping sequence elements reduce the overall duration of the pulse sequences, theprincipal benefit derives from a reduction in the number of 180° pulses. Two versions of thetechnique are presented: a 3D (H)N(COCA)NH experiment that correlates 15N(i),1H(i-1), and 15N(i-1) spins, and a 3D HN(COCA)NH experiment that correlates 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins by simultaneously encoding the 1H(i) and 15N(i) chemical shiftsduring the t1 evolution period. The methods are demonstrated on a 13C/15N-enriched sampleof the protein ubiquitin and are easily adapted for application to 2H/13C/15N-enrichedproteins.     

Assignment of the backbone carbonyl resonances in 15N-labelled proteins with 13C at natural abundance by a 2D triple-resonance correlation technique

Summary A 2D NMR experiment for assignment of backbone carbon resonances in small and medium-sized ¹⁵N-labelled proteins with ¹³C at natural abundance is presented. The experiment is a two-dimensional variant of the HNCO triple-resonance experiment and is demonstrated by application to a 6 kDa protein at relatively low concentration (2 mM) and temperature (30°C). The experiment is particularly suitable for assignment of carbonyl resonances.     

One simple step in the identification of the cofactors signals, one giant leap for the solution structure determination of multiheme proteins

Multiheme proteins play major roles in various biological systems. Structural information on these systems in solution is crucial to understand their functional mechanisms. However, the presence of numerous proton-containing groups in the heme cofactors and the magnetic properties of the heme iron, in particular in the oxidised state, complicates significantly the assignment of the NMR signals. Consequently, the multiheme proteins superfamily is extremely under-represented in structural databases, which constitutes a severe bottleneck in the elucidation of their structural-functional relationships. In this work, we present a strategy that simplifies the assignment of the NMR signals in multiheme proteins and, concomitantly, their solution structure determination, using the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens as a model. Cost-effective isotopic labeling was used to double label (¹³C/¹⁵N) the protein in its polypeptide chain, with the correct folding and heme post-translational modifications. The combined analysis of ¹H-¹³C HSQC NMR spectra obtained for labeled and unlabeled samples of PpcA allowed a straight discrimination between the heme cofactors and the polypeptide chain signals and their confident assignment. The results presented here will be the foundations to assist solution structure determination of multiheme proteins, which are still very scarce in the literature.     

Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cgamma-Hgamma/Cbeta-Hbeta residual dipolar couplings: application to the 34-kDa enzyme IIA(chitobiose)

An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a ¹⁵N/¹³C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ(i)–H_N(i) and Hγ(i)–H_N(i+1) NOEs in a 3D ¹⁵N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine ¹³C–¹H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA^Chitobiose, a 34 kDa homotrimeric phosphotransferase protein.     

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

Summary Two new 3D ¹H-¹⁵N-¹³C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the ²J_NC coupling to establish the sequential assignment of backbone NH and ¹⁵N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly ¹⁵N- and ¹³C-labelled ribonuclease T1.     

Optimized set of two-dimensional experiments for fast sequential assignment,secondary structure determination,and backbone fold validation of 13C/15N-labelled proteins

NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional ¹H-¹⁵N HSQC-like spectra. The ¹H-¹⁵N correlation peaks are frequency shifted by an amount ± _X along the ¹⁵N dimension, where _X is the C, C, or H frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.     

Towards high-resolution solid-state NMR on large uniformly 15N- and [13C,15N]-labeled membrane proteins in oriented lipid bilayers

Based on exact numerical simulations, taking into account isotropic and conformation-dependent anisotropic nuclear spin interactions, we systematically analyse the prospects for high-resolution solid-state NMR on large isotope-labeled membrane proteins macroscopically oriented in phospholipid bilayers. Using the known X-ray structures of rhodopsin and porin as models for large membrane proteins with typical -helical and -barrel structural motifs, the analysis considers all possible one- to six-dimensional spectra comprised of frequency dimensions with evolution under any combination of amide ¹H, amide ¹⁵N, and carbonyl ¹³C chemical shifts as well as ¹H-¹⁵N dipole-dipole couplings. Under consideration of typical nuclear spin interaction and experimental line-shape parameters, the analysis provides new insight into the resolution capability and orientation-dependent transfer efficiency of existing experiments as well as guidelines as to improved experimental approaches for the study of large uniformly ¹⁵N- and [¹³C,¹⁵N]-labeled membrane proteins. On basis of these results and numerical optimizations of coherence-transfer efficiencies, we propose several new high-resolution experiments for sequential protein backbone assignment and structure determination.     

Improved resolution in three-dimensional constant-time triple resonance NMR spectroscopy of proteins

Summary Two new protocols for the three-dimensional, triple resonance, constant-time HCA(CO)N NMR experiment are presented that significantly increase the experimental resolution attainable in the C frequency dimension. Experimental verification of the new experiments is provided by spectra of the IIA domain of glucose permease fromBacillus subtilis.     

Overcoming the problems associated with poor spectra quality of the protein kinase Byr2 using residual dipolar couplings

For the Ras-binding domain of the protein kinase Byr2, only a limited number of NOE contacts could be initially assigned unambiguously, as the quality of the NOESY spectra was too poor. However, the use of residual (1)H-(15)N dipolar couplings in the beginning of the structure determination process allows to overcome this problem. We used a three-step recipe for this procedure. A previously unknown structure could be calculated reasonably well with only a limited number of unambiguously assigned NOE contacts.     

Resolution of the 1H-1H NOE spectrum of RNA into three dimensions using 15N-1H two-bond couplings

The feasibility of using two-bond ¹⁵N-¹H couplings to resolve the ¹H-¹H nuclear Overhauser effect spectrum of RNA into a third dimension was investigated, using the 36-nucleotide gene 32 messenger RNA pseudoknot of bacteriophage T2 as an example. The two-bond ¹⁵N-¹H couplings present in adenosine and guanosine were found to be suitable for generating a three-dimensional ¹H-¹H-¹⁵N NOESY-HSQC spectrum with reasonably good sensitivity, as well as favorable chemical shift dispersion in the nitrogen dimension. The described NMR experiment provides a tool that can be used to complement other heteronuclear methods in the analysis of RNA structure.     

Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of ¹³C and ¹⁵ N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to ¹³C and ¹⁵N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the ¹³C and ¹⁵N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields N_i-C_i–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a ¹³C spin diffusion experiment, which exhibits ¹³C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive ¹³ C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive ¹³C labeling approach.     

Measurement of small scalar and dipolar couplings in purine and pyrimidine bases*

A suite of spin-state-selective excitation (S³E) NMR experiments for the measurements of small one-bond (¹³C-¹³C, ¹⁵N-¹³C) and two-bond (¹H-¹³C, ¹H-¹⁵N) coupling constants in ¹³C,¹⁵N labeled purine and pyrimidine bases is presented. The incorporation of band-selective shaped pulses, elimination of the cross talk between and sub-spectra, and accuracy and precision of the proposed approach are discussed. Merits of using S³E rather than /-half-filter are demonstrated using results obtained on isotopically labeled DNA oligonucleotides.     

Accurate measurement of residual dipolar couplings in anisotropic phase

The determination of residual dipolar couplings (RDCs) by quantitative J spectroscopy methods such as Heteronuclear Single Quantum Correlation with Phase Encoded Coupling (HSQC-PEC) is prone to systematic errors that may be caused by differential attenuation during the conversion of orthogonal density operator components into observable terms. The attenuation may be caused by miscalibration of radio-frequency pulses and by relaxation effects. A simple method is presented that allows one to remove most of these systematic errors without losses in sensitivity or resolution.     

3D 13C-15N-heteronuclear two-spin coherence spectroscopy for polypeptide backbone assignments in 13C-15N-double-labeled proteins

Summary The pulse sequence of a new constant-time 3D triple-resonance experiment, ct-HA[CAN]HN, is presented. This experiment delineates exclusively scalar connectivities and uses ¹³C–¹⁵N heteronuclear two-spin coherence to overlay the chemical shift evolution periods of the ¹³C and ¹⁵N nuclei, thereby providing the four resonance frequencies of the -proton, the -carbon, the amide nitrogen, and the amide proton of a given amino acid residue in three dimensions. This experiment promises to be a valid alternative to 4D experiments, providing the same information on intraresidue polypeptide backbone connectivities in ¹³C-¹⁵N-double-labeled proteins.Abbreviations 3D, 4D three-dimensional, four-dimensional - TPPI time-proportional phase incrementation - ct constant-time - rf radiofrequency - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - glutaredoxin(C14S) mutant E. coli glutaredoxin with the cysteine at position 14 replaced by serine